Protein tyrosine phosphatases (PTP's) are a class of enzymes, which just recently have been linked to various diseases including cancer, cardiovascular, immunological, infectious, neurological, and metabolic diseases (Tautz, L. et al., (2006) Targeting the PTPome in human disease. Ex. Op Ther Tar, 10, 157-177). Vaccinia H1-related (VHR) phosphatase is a dual-specificity phosphatase, which was cloned on the basis of sequence homology with the first identified dual-specificity protein phosphatase, the Vaccinia virus H1 open reading frame (Ishibashi, T. et al., (1992) Expression cloning of a human dual-specificity phosphatase. Proc Natl Acad Sci USA 89, 12170-4). VHR is a small enzyme with only 185 amino acids (Mr 21 kDa), and it doesn't comprise any apparent targeting domain or docking site. The crystal structure of VHR has been solved, identifying a shallow active site that allows VHR to act on both phospho-tyrosine (pTyr) and phospho-threonine (pThr) (Yuvniyama, J. et al., (1996) Crystal structure of the dual specificity protein phosphatase VHR, Science 172, 1328-31). VHR has been reported to dephosphorylate the mitogen-activated protein kinases (MAP kinases) Erk and Jnk, but not p38 (Alonso, A. et al., (2001) Inhibitory role for dual specificity phosphatase VHR in T cell antigen receptor and CD28-induced Erk and Jnk activation. J Biol Chem 276, 4766-71; Todd, J. L. et al., (2002) Dual-specificity protein tyrosine phosphatase VHR down-regulates c-Jun N-terminal knase (NK), Oncogene 21, 2573-83, Todd, J. L. et al., (1999) Extracellular regulated kinases (ERK) 1 and ERK2 are authentic substrates for the dual-specificity protein-tyrosine phosphatase VHR. A novel role in down-regulating the ERK pathway. J Bio Chem 274, 13271-80). MAP kinases mediate major signaling pathways triggered by extracellular growth factor, stress, and cytokines (Waskiewicz, A. J. et al., (1995) Mitogen and stress response pathways: MAP kinase cascades and phosphatase regulation in mammals and yeast. Curr Opin Cell Bio 7, 798-805) and regulate cell differentiation, proliferation and apoptosis (Robinson, M. J. et al., (1997) Mitogen-activated protein kinase pathways. Curr Opin Cell Biol 9, 180-6; Ip, Y. T. et al., (1998) Signal transduction by the c-Jun N-terminal kinase (JNK)—from inflammation to development. Curr Opin Cell Biol 10, 205-19).
MAP-kinases are activated by phosphorylation at a Thr-X-Tyr motif in the activation loop (Canagarajah, B. J. et al., (1997) Activation mechanism of MAP kinase ERK2 by dual phosphorylation. Cell 90, 859-69) and then phosphorylate their cellular substrates, including many transcription factors required for the expression of cell cycle regulatory genes, such as cyclins that regulate cyclin-dependent kinases. The inactivation of MAP kinases is catalyzed by phosphatases that dephosphorylate the pThr and/or pTyr in the activation loop, such as VHR.
VHR is activated by phosphorylation at Y138 by the ZAP-70 tyrosine kinase (Alonso, A. et al., (2003) Tyrosine phosphorylation of VHR phosphatase by ZAP-70. Nat. Immunol. 4, 44-8) and probably other kinases. Unlike many other MAP kinase phosphatases (MKP's), VHR expression is not induced in response to activation of MAP kinases (Alonso, A. et al., (2001) Inhibitory role for dual specificity phosphatase VHR in T cell antigen receptor and CD28-induced Erk and Jnk activation. J Biol Chem 276, 4766-71), but is instead connected to cell cycle progression (Rahmouni, S. et al., (2006) Loss of VHR causes cell-cycle arrest and senescence. Nat Cell Biol. 8, 524-531). Using RNA interference to knock down endogenous VHR in HeLa carcinoma cells, cell cycle is arrested at G1 to S and G2 to M transitions and cells show signs of senescence, suggesting that VHR inhibition may be a useful approach to halt the growth of cancer cells. Loss of VHR decreases the expression of cell cycle regulators CDC2, CDK2 and CDK4, matching the results for cells entering senescence (Stein, G. H. et al., (1991) Senescent cells fail to express cdc2, cycA, and cycB in response to mitogen stimulation. Proc Natl Acad Sci USA 88, 11012-6), whereas the most up-regulated gene in VHR knock down is the CDK inhibitor p21Cip-waf1. In synchronized cells, VHR is hardly detectable in G1 phase, and then slowly increases when cells go through cell cycle and peaked in M phase. When cells reach the next G1 phase, VHR levels are quickly back to minimal. When cells are treated with protein synthesis inhibitor cycloheximide, the half-life of VHR in G1 phase is much shortened, compared to other phases.
When VHR is knocked down there is a strong activation of Erk and Jnk, the only two substrates identified for VHR. Without VHR, activities of both Erk and Jnk are highly elevated after activation and there is a clear increase of the basal Erk activity (Rahmouni, ibid.). There have been several reports that prolonged activation of MAP kinase pathway results in cell cycle arrest and cell senescence (Woods, D. et al., (1997) Raf-induced proliferation or cell cycle arrest is determined by the level of Raf activity with arrest mediated by p21Cip1. Mol Cell Biol 17, 5598-611; Sewing, A. et al., (1997) High-intensity Raf signal causes cell cycle arrest mediated by p21Cip1. Mol Cell Biol 17, 5588-97; Serrano, M. et al., (1997) Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p161NK4a. Cell 88, 593-602; Pumiglia, K. M. et al., (1997) Cell cycle arrest mediated by the MEK/mitogen-activated protein kinase pathway. Proc Natl Acad Sci USA 94, 4448-52; Wang, W. et al. (2002) Sequential activation of the MEK-extracellular signal-regulated kinase and MKK3/6-p38 mitogen-activated protein kinase pathways mediates oncogenic ras-induced premature senescence. Mol Cell Biol 22, 3389-403). Elevated Erk is directly responsible for M-phase arrest, and inactivation of Erk is required to exit M-phase (Chau, A. S. et al., (1999) Inactivation of p42 mitogen-activated protein kinase is required for exit from M-phase after cyclin destruction. J Biol Chem 274, 32085-90). Also, Jnk activation has been linked to G1-phase arrest in response to growth inhibitory stimuli (Tchou, W. W. et al., (1999) Role of c-Jun N-terminal kinase 1 (JNK-1) in cell cycle checkpoint activated by the protease inhibitor N-acetyl-leucinyl-leucinyl-norleucinal. Oncogene 18, 6974-80; Grosch, S. et al., (2003) Activation of c-Jun-N-terminal-kinase is crucial for the induction of a cell cycle arrest in human colon carcinoma cells caused by flurbiprofen enantiomers. Faseb J 17, 1316-8). Stress has been shown to activate Jnk and to induce p53 and 21Cip-waf1 expression (Xue, Y. et al., (2003) Association of JNK1 with p21waf1 and p53: modulation of JNK1 activity. Mol Carcinog 36, 38-44). Jnk has been confirmed to be physically interacting with p53 and p21Cip-waf1 (Xue ibid.). Loss of VHR induced cell cycle arrest is dependent on the hyperactivation of Erk and Jnk, with Erk responsible for G2-M arrest and Jnk responsible of the G1-S arrest (Rahmouni ibid.).
The first VHR small molecule inhibitor to be described was the tetronic acid derivative RK-682 which was isolated from a Streptomyces strain as a PTP inhibitor in a microbial metabolites screening (Hamaguchi, T. et al., (1995) Rk-682, a potent inhibitor of tyrosine phosphatase, arrested the mammalian cell cycle progression at G1 phase. FEBS Lett. 372, 54-58). In vitro, RK-682 inhibited cell cycle progression of Ball-1 cells, arresting them at the G1/S cell cycle phase transition. However, RK-682 was found to have several other inhibitory activities, including phospholipase A2 inhibition (Shinagawa, S. et al., (1993) Tetronic acid derivatives, its manufacturing methods and uses. Japan. Kokai Tokyo Koho, JP 05-43568, 1-26), HIV-1 protease inhibition (Roggo, B. E. et al., (1994) 3-alkanoyl-5-hydroxymethyl tetronic acid homologues and resistomycin: new inhibitors of HIV-1 protease. I. Fermentation, isolation and biological activity. J Antibiot. (Tokyo) 47, 136-42; Roggo, B. E. et al., (1994) 3-alkanoyl-5-hydroxymethyl tetronic acid homologues: new inhibitors of HIV-1 protease. II. Structure determination. J. Antibiot. (Tokyo) 47, 143-7.) and heparanase inhibition (Ishida, K. et al., (2004) Exploitation of heparanase inhibitors from microbial metabolites using an efficient visual screening system. J Antibiot. 57, 136-42).